FIG. 6.

Mouse Dab1RFP fusion is tyrosine-phosphorylated when expressed in Rat-2 cells and fly eyes. (A) Cell lysates from Rat-2 fibroblasts transfected with Dab1GFP (lanes 1 and 4), Dab1-wt (lanes 2 and 5), or Dab1RFP (lanes 3 and 6) were immunoprecipitated with anti-Dab1 C-terminal antibody. Western blotting with anti-Dab1 antibody (lanes 1 to 3) showed that comparable levels of Dab1 were expressed, but in the antiphosphotyrosine Western blot (lanes 3 to 6), only Dab1RFP (lane 6) was detected. (B) Expression of Dab1RFP and Dab1RFP-5F was detected in total cell lysates of flies with the genotypes UAS-Dab1RFP/GMR-GAL4 (lane 1), UAS-Dab1RFP-5F/GMR-GAL4 (lane 2), and UAS-Dab1RFP-5F;UAS-Dab1RFP-5F/GMR-GAL4 (lane 3) to compare the levels of expression of the wild-type and Dab1-5F mutant flies as adults. Expression of both of these protein products was also observed by Western blot in wandering third-instar larvae and pupae (not shown). Lysates from UAS-Dab1RFP/GMR-GAL4 (lane 4) and UAS-Dab1RFP-5F/GMR-GAL4 (lane 5) flies were immunoprecipitated with anti-Dab1 antibody (Chemicon) and Western blotted for antiphosphotyrosine (4G10; Upstate Biotechnology), showing that the Dab1RFP fusion but not the Dab1RFP-5F protein is tyrosine phosphorylated when expressed in the adult fly. The same was observed for wandering third-instar larvae and pupae (not shown).